We have used site-directed spin labeling to prove the active site of phosphoribulokinase, an enzyme in the Calvin cycle that catalyzes the transfer of a phosphoryl group from ATP to ribulose 5-phosohate. To this end, we have generated two single cysteine mutants A16C and k53C by site directed mutagenesis, in the active site of PRK. The two mutants were characterized for activity and also checked for structural perturbation using filtration, CD and titration of the active site with the spin labeled ATP probe, ATPSAP. The mutants show no noticeable deviation from Wild Type. They were then spin labeled with MTSSL and their spectra recorded in the presence and absence of ATP and the allosteric effector NADH. SL-K53C spectrum was not sensitive to ATP or NADH while the A16C spectrum was sensitive to ATP. The latter is expected due to the fact that A16 is a residue in the P-loop which is known to be the nucleotide binding site. Experiments are in progress on the double mutant A16C/K53C to determine distances in the active site.